Some blood vessels don't show up on circulatory system diagrams, or even in the plain light of a laboratory lamp. These are microscopic, forming networks at the ends of the system, feeding living tissue with nutrients and carrying off wastes.

Because these blood vessels support the vitality of all organs and muscles, the mystery of how they're put together is central for scientists working on producing replacement body parts from the raw material of cells to treat damaged organs. Researchers have been working on elements of the riddle for years.

Scientists at the Johns Hopkins University recently advanced the effort by simplifying the method of creating these microvascular networks in the laboratory and demonstrating that they work in living animals. The development, published this summer in the Proceedings of the National Academy of Sciences, could bring these life-supporting vessels a step closer to clinical use, for treating wounds, perhaps, or diabetes, stroke and heart disease.

"Many organs can benefit from vascular support," giving the project potentially broad application, said Sharon Gerecht, an associate professor in the Hopkins department of chemical and biomolecular engineering who has been leading the team of faculty members and graduate students in the effort. The paper in the Proceedings of the National Academy of Sciences referred to the "vast ramifications for vascular construction" and medical treatment focusing on regenerating damaged tissue.

A key moment came about 18 months ago. The scientists saw that stem cells created from adult human blood cells — called "pluripotent" stem cells, meaning they can produce an array of tissues — had developed using a new, more efficient method, forming vessels within a three-dimensional jelly-like support called hydrogel.

"When you ask me the 'eureka,' it's this," said Gerecht, pointing to an image on a computer screen taken of a 10-magnification view of the vessels. That image, she said, represents about 10 years of work.

Dyed lilac color and in a blown-up view, the image might be a stylized watercolor of a tangle of rose branches, some closer to the picture surface than others. The vessels appear as pale tendrils, their slightly darker centers marking the channels that would carry blood.

In the more than 20-year history of tissue engineering in the United States, it's not the first time that scientists have created blood vessels in the laboratory using stem cells. It is, however, the first time that it's been done with this method, which selects more quickly for the two types of cells needed to create blood vessels: endothelial cells that create the vessel lining, and pericytes that wrap the cells and support the vessel.

Previous research has taken other, more cumbersome approaches: for instance, growing vessels using endothelial cells, then building the supporting pericytes in a second step.

In the method fashioned by doctoral student Sravanti Kusuma, the two types of cells develop simultaneously. She said the approach saves time that would be spent sorting through an array of cell types to find those two, or growing separate batches of endothelial and pericyte cells.

Yunling Gao, program director for the National Heart, Lung and Blood Institute, said in an email that this approach sets this work apart.

"This is the first report to demonstrate that pluripotent stem cells can be induced to differentiate specifically just into the two cell types needed to build new blood vessels," said Gao, whose agency is part of the National Institutes of Health and contributed grant money to the research. Also contributing were the American Heart Association and the National Science Foundation.

Rosemarie Hunziker, program director of the National Institute of Biomedical Imaging and Bioengineering, also part of the NIH, said the Gerecht group is making this process "more simple and elegant," and more accessible.

"It's one process as opposed to two processes. That is very powerful," said Hunziker. "It's hugely important."

Researchers implanted the vessels in the hydrogel into mice, where they became integrated with the animal's own blood vessels. An injection of green fluorescent liquid into the tails of the mice showed that the vessels were carrying blood.

Gerecht's team has tested the vessels in live mice for up to two weeks, but she said another group has found that vascular networks developed from stem cells can last for more than nine months in mice.

Gerecht emphasized that this would not have been possible without the work of Linzhao Cheng of Hopkins' Institute for Cell Engineering, who produced the human pluripotent stem cells by reverting the patient's blood cells to an earlier form. In that form, they were induced by chemical cues to create the endothelial and pericyte cells.

Working backward from adult cells allows the possibility of making customized vessels from a patient's own blood. That would avoid having to use drugs to dampen immune system response to foreign tissue.

But that's getting ahead of the story. There are still many questions left to answer about these blood vessels, said Gerecht and Hunziker.

How do the vessels function in treating disease? How long can the vessels be maintained in the hydrogel? What is the extent of physical cues that causes the cells to differentiate into the blood vessel cells? How would the vessels function to support a complex organ, such as the kidney?

For now, the researchers will be working with a new local venture that wants to apply their techniques to create wound-healing products for the commercial market. In the long run, they may have taken a step toward the tissue engineers' long-held goal of making replacement parts tailored to the patient.

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